Mechanistic insights into SARS-CoV-2 spike protein induction of the chemokine CXCL10

During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of cytokines can lead to severe COVID-19. Previous studies suggest that the spike protein (S) of SARS-CoV-2 can elicit cytokine production via the transcription factor NF-κB and the toll-like receptors (TLRs). In this study, we found that: (i) S and the S2 subunit induce CXCL10, a chemokine implicated in severe COVID-19, gene expression by human macrophage cells (THP-1); (ii) a glycogen synthase kinase-3 inhibitor attenuates this induction; (iii) S and S2 do not activate NF-κB but do activate the transcription factor IRF; (iv) S and S2 do not require TLR2 to elicit CXCL10 production or activate IRF; and (v) S and S2 elicit CXCL10 production by peripheral blood mononuclear cells (PBMCs). We also discovered that the cellular response, or lack thereof, to S and S2 is a function of the recombinant S and S2 used. While such a finding raises the possibility of confounding LPS contamination, we offer evidence that potential contaminating LPS does not underly induced increases in CXCL10. Combined, these results provide insights into the complex immune response to SARS-CoV-2 and suggest possible therapeutic targets for severe COVID-19.

of the main text.Protein arrays exposed to supernatants from THP-1 macrophages treated with AS and AS2 proteins +/-COB-187 revealed: supernatants from untreated THP-1 macrophages had little, if any, CXCL10 (location 4, panel a); AS or AS2 protein alone and in the presence of 1% DMSO (carrier control for COB-187) increased CXCL10 protein levels (location 4 panels b,c,d,e); COB-187 appeared to abolish the AS-and AS2-induced increase in CXCL10 protein levels (location 4 panels f,g).Note that AS2 appeared to increase levels of 3 other proteins (locations 1,2,3; panels c) that were dramatically reduced by COB-187 (locations 1,2,3; panel g) but unaffected by 1% DMSO (locations 1,2,3; panel e).The map provides the correspondence between the number on the array and the cytokine present at that location.Each cytokine is in duplicate (two dots per location).The two dark pairs of dots on the left side of each array and the dark pair of dots on the right side of the array are positive controls.S4.Replicate experiments for Fig. 7. Mean and SD were determined from duplicates for each condition within each replicate.Compared to media control via t-test: NS indicates not significant; LTC indicates lower than control: #p<0.05;*p<0.025;* * p< 0.01; %p<0.0083(the cutoff for significance with a Bonferroni correction involving 6 comparisons); and ***p<0.001.Threshold for significant difference is p<0.05 without Bonferroni correction.Threshold for significant difference is p<0.025(Figs.7a,b) and p<0.0083 (Fig. 7c) with Bonferroni correction.AS and AS2 were at 25 nM.Replicate 1 from each set is presented in Fig. 7.Note that the positive controls (AS, AS2) for the replicates in Fig. 7c provide two additional replicates for 7a.
Figure S1.S and S2 treatment increase CXCL10 protein levels and this increase is attenuated by COB-187.This figure is a replicate experiment of the representative figure presented in Fig.1of the main text.Protein arrays exposed to supernatants from THP-1 macrophages treated with AS and AS2 proteins +/-COB-187 revealed: supernatants from untreated THP-1 macrophages had little, if any, CXCL10 (location 4, panel a); AS or AS2 protein alone and in the presence of 1% DMSO (carrier control for COB-187) increased CXCL10 protein levels (location 4 panels b,c,d,e); COB-187 appeared to abolish the AS-and AS2-induced increase in CXCL10 protein levels (location 4 panels f,g).Note that AS2 appeared to increase levels of 3 other proteins (locations 1,2,3; panels c) that were dramatically reduced by COB-187 (locations 1,2,3; panel g) but unaffected by 1% DMSO (locations 1,2,3; panel e).The map provides the correspondence between the number on the array and the cytokine present at that location.Each cytokine is in duplicate (two dots per location).The two dark pairs of dots on the left side of each array and the dark pair of dots on the right side of the array are positive controls.

Figure S2 .
Figure S2.THP-1 and DRTHP1, but not KO-DRTHP1, macrophages have surface expressed TLR2 protein.Further replicates of the representative data presented in Figs. 3 of the main text.Flow cytometric analysis revealed that a mAb to TLR2 bound to THP-1 (a,b) and DRTHP1 (c) macrophages (red histograms) to a greater extent than an isotype matched control (blue histograms) suggesting that these cells express surface TLR2 protein.(d) Flow cytometric analysis revealed that a mAb to TLR2 did not bind to KO-DRTHP1 macrophages (red histogram) to a greater extent than an isotype matched control (blue histogram) suggesting that KO-DRTHP1 macrophages do not express surface TLR2 protein.

Figure S3 .
Figure S3.AS and AS2 do not appear to activate NF-κB in THP-1 macrophages.The following two pages contain the original blots from which Figs. 6c and 6d were generated.

Table S1 .
Replicate experiments for Fig.4.Mean and SD determined from duplicates for each condition within a replicate.Compared to media control via t-test: * p <0.025; * * p< 0.01; and ***p<0.001.Threshold for significant difference is p<0.05 and p<0.025 without and with Bonferroni correction, respectively.AS and AS2 were at 25 nM.Replicate 1 from each set is presented in Fig.4.

Table S2 .
Replicate experiments for Fig.5.Mean and SD were determined from duplicates for each condition within each replicate.Compared to media control via t-test: NS indicates not significant; LTC indicates lower than control; #p<0.05;**p< 0.01; and ***p<0.001.Threshold for significant difference is p<0.05 and p<0.025 without and with Bonferroni correction, respetively.A Bonferroni correction is not needed for Figs.5c and 5d since only a single comparison was made.AS and AS2 were at 25 nM.Pam3CSK4 was at 1000 ng/ml.Replicate 1 from each set is presented in Fig.5.

Table S3 .
Replicate experiments for Fig.6a,b.Mean and SD determined from duplicates for each condition within a replicate.For 6a: Compared to DMSO via two sample t-test.For 6b, compared to 100% via one sample t-test.NS indicates not significant; #p<0.05.Threshold for significant difference is p<0.05.A Bonferroni correction was not used for the one sample t-test in 6b.AS and AS2 were at 25 nM.Replicate 1 is presented in Fig.6a.

Table S5 .
Replicate experiments for Fig.8.Mean and SD determined from duplicates for each condition within a replicate; quadruplicates used for RS2 8b, Rep 1.Compared to media matched control for 8a, 8b, 8c via t-test; compared to same condition with PB for 8d via t-test: NS indicates not significant; NP indicates not performed; LTC indicates lower than control; #p<0.05;**p< 0.01; and ***p<0.001.Threshold for significant difference is p<0.05 without Bonferroni correction and p<0.025 with Bonferroni corretion.Concentration of spike proteins was 25 nM.Replicate 1 from each set (8a, 8b, 8c) and the 1 EU/ml condition (8d) is presented in Fig.8.